Biology subjects

Cate, J.

Publications and source records attributed to Cate, J..

11 recordsLinked to original sources

bioRxiv2026

eIF3 interactions with miRNAs involved in translational control of early T cell activation

Early T cell activation induces extensive remodeling of the cellular transcriptome and proteome. We previously showed using a transcriptome-wide crosslinking approach that human translation initiation factor eIF3 directly interacts with a select set of immune-related mRNAs shortly after T cell activation. We also found that eIF3 binding to the 3'-untranslated regions (3'-UTRs) of the TCRA and TCRB mRNAs encoding the T cell receptor alpha and beta subunits dynamically regulates a burst in their translation. MicroRNAs (miRNAs) add an additional layer of regulation by fine-tuning both mRNA and protein expression. Although miRNA expression is dynamically regulated during T cell activation, how miRNAs interact with core regulatory pathways in primary T cells remains poorly understood. Here, we reexamined the eIF3-RNA crosslinking experiments in Jurkat cells and probed eIF3 function to investigate miRNA-mediated translational regulation during T cell activation. We found that eIF3 interacts with multiple mature miRNAs in activated Jurkat cells, including members of the miR-17[~]92 cluster. These interactions also occur in primary T cells, as shown by RNA immunoprecipitation followed by qPCR (RIP-qPCR). Knocking out the miR-17[~]92 cluster led to a delay in ILR2A (CD25) cell surface expression in Jurkat cells and reduced activation-associated cell size increase in primary T cells during early T cell activation. In parallel, we used mass spectrometry to identify eIF3-interacting factors in activated primary T cells, and surprisingly found no evidence of Argonaute binding. Together, these findings provide evidence that eIF3-miRNA interactions may play an unappreciated role in translational control during early T cell activation.

Molecular Biology↗

bioRxiv2020

crispr2vec: Machine Learning Model Predicts Off-Target Cuts of CRISPR systems

1Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Cas systems have revolutionized gene editing, with applications in therapeutics, diagnostics, agriculture, and developing disease models. However, CRISPR-Cas suffers from off-target effects -- unintended genetic modifications in the genome that arise from its use. In this work, we present crispr2vec: a deep metric learning approach for embedding CRISPR single guide RNA (sgRNA) sequences and predicting off-target cuts. Given a fixed target sequence, we show that our learned embedding yields a faithful representation of potential off-targets. We present a new triplet sampling strategy specifically for CRISPR sequences that improves the quality of our embedding. We show the resulting embedding generalizes across different off-target cut detection assays. Finally, we demonstrate the superiority of our deep metric learning method in its ability to predict off-target cuts compared to previous literature in cross fold validation across different datasets for both seen and unseen sgRNAs.

synthetic biology↗

bioRxiv2018

PTBP1 mRNA isoforms and regulation of their translation

Polypyrimidine tract-binding proteins (PTBPs) are RNA binding proteins that regulate a number of post-transcriptional events. Human PTBP1 transits between the nucleus and cytoplasm and is thought to regulate RNA processes in both. However, information about PTBP1 mRNA isoforms and regulation of PTPB1 expression remain incomplete. Here we mapped the major PTBP1 mRNA isoforms in HEK293T cells, and identified alternative 5 and 3untranslated regions (5 UTRs, 3 UTRs) as well as alternative splicing patterns in the protein coding region. We also assessed how the observed PTBP1 mRNA isoforms contribute to PTBP1 expression in different phases of the cell cycle. Previously, PTBP1 mRNAs were shown to crosslink to eukaryotic translation initiation factor 3 (eIF3). We find that eIF3 binds differently to each PTBP1 mRNA isoform in a cell cycle-dependent manner. We also observe a strong correlation between eIF3 binding to PTBP1 mRNAs and repression of PTBP1 levels during the S phase of the cell cycle. Our results provide evidence of translational regulation of PTBP1 protein levels during the cell cycle, which may affect downstream regulation of alternative splicing and translation mediated by PTBP1 protein isoforms.

molecular biology↗

bioRxiv2018

Cellular response to small molecules that selectively stall protein synthesis by the ribosome

Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease.

systems biology↗

bioRxiv2018

Kluyveromyces marxianus as a robust synthetic biology platform host

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineer the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits- thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.

synthetic biology↗

bioRxiv2018

Cancer-associated mRNAs regulated by the Helix-Loop-Helix motif of human EIF3A

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5-UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

cancer biology↗

bioRxiv2018

Orthogonal regulation of ferritin translation by human eIF3 and the iron response proteins

A central problem in human biology remains the discovery of causal molecular links between mutations identified in genome-wide association studies (GWAS) and their corresponding disease traits. This challenge is magnified for variants residing in non-coding regions of the genome. Single-nucleotide polymorphisms (SNPs) in the 5 untranslated region (5-UTR) of the ferritin light chain (FTL) gene that cause hyperferritinemia are thought to disrupt translation repression by altering iron regulatory protein (IRP) interactions with the FTL mRNA 5-UTR. Here, we show that human eukaryotic translation initiation factor 3 (eIF3) acts as a distinct repressor of FTL mRNA translation, and eIF3-mediated FTL repression is disrupted by a subset of SNPs in FTL that cause hyperferritinemia. These results identify a direct role for eIF3-mediated translational control in a specific human disease.

molecular biology↗

bioRxiv2018

Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

Small molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

biophysics↗

bioRxiv2017

Programmable RNA recognition using a CRISPR-associated Argonaute

Argonaute proteins (Agos) are present in all domains of life. While the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci and use non-canonical 5-hydroxyled guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5 nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5-BrdU modified gRNAs, we mapped the seed region of the gRNA, and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the 6th and 7th positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific Adenosine to Inosine edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5-BrdU modified gRNAs can be used as a highly-specific RNA-targeting platform to probe RNA biology.\n\nSIGNIFICANCEArgonaute proteins are present in bacteria, archaea and eukaryotes. They play an important role in a wide range of biological processes, from transcriptional and translational gene expression regulation to defense against viruses and silencing of mobile genetic elements. Here we present mechanistic insights into the interactions of the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) with its guide RNA (gRNA) and RNA substrates. By modifying the 5-nucleotide of the gRNA, we demonstrate that MpAgo-gRNA complexes (RNPs) are easily programmable, have high affinity to fully complementary RNA substrates, and can discriminate by over 300 fold between substrates that differ by only a single nucleotide. These MpAgo RNPs should be useful for probing endogenous RNAs in living cells.

biochemistry↗

bioRxiv2017

Role for Ribosome-Associated Complex and Stress-Seventy subfamily B (RAC-Ssb) in integral membrane protein translation

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle (SRP), which recognizes a hydrophobic signal sequence near the protein N-terminus. Proper folding of these proteins is monitored by the unfolded protein response, and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix-the signal sequence recognized by SRP-is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex (RAC) and Stress-Seventy Subfamily B (Ssb) chaperones. We also show that quality control of integral membrane proteins by RAC-Ssb couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.

molecular biology↗

bioRxiv2016

A compound that directly and selectively stalls PCSK9 translation

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a key role in regulating the levels of plasma low density lipoprotein cholesterol (LDL-C). Here we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome, which allows small molecules to specifically block translation of individual transcripts.\n\nOne Sentence SummaryA small-molecule PCSK9 inhibitor targets the human ribosome and selectively prevents PCSK9 synthesis.

systems biology↗